PRIMERA ENCUESTA EXPLORATORIA

The perceived contributions of glutaraldehyde treatment to BHV failure have lead to a search for suitable alternatives. Alterations to glutaraldehyde fixation methods as well as replacements have been pursued in recent years. Modifications have ranged from changes as simple as using higher concentrations to more involved strategies that reduce tissue reactivity, block residual aldehyde groups, or incorporate into the crosslinks [78,104]. Some non-glutaraldehyde tissue fixatives that have been investigated include: epoxy compounds, such as triglycidalamine [105]; carbodiimides [103,114-120]; acylazides [103]; poly-glycidyl ethers [106]; reuterin [107]; genipin [108]; glycerol [103]; sodium metaperiodate [88]; diisocyanates [103]; and dye-mediated photooxidation

[109-112]. Many of these fixatives have yielded promising results through preliminary testing, but there has been minimal conversion into clinical use.

The primary focus of many studies involving these new chemistries has been on the reduction of calcification, though there are additional benefits that may also accompany the nature of this strategy. Possible benefits include: potential enhancement to tissue stability, improved biomechanics, and reduction of fixative toxicity. However, the use of entirely new fixation chemistries also risks providing inadequate tissue durability as well as stimulating unknown host responses. BHVs crosslinked by dye- mediated photooxidation and carbodiimides have progressed to clinical trials; however, neither has yet further advanced to regular clinical use. Both of these particularly promising compounds do not become incorporated into the tissue, but merely catalyze the formation of covalent bonds within the tissue. This particular characteristic all but ensures reduced toxicity and early results have demonstrated reduced calcification in tissues crosslinked by each treatment [109-111,116-119]. The use of these new crosslinking methods, along with proper valve construction, may improve the average lifetime of BHVs representing the next evolution in their design.

1.7.1. Dye-mediated photooxidation

Fixation processes using dye-mediated photooxidation are fairly simple and include incubation in the photooxidative dye of interest, followed by exposure to specific wavelengths of light in an aerobic environment. However, the mechanisms behind this method of crosslink formation are not fully understood [111-112]. After light absorption by the dye, excitation initiates the crosslinking response that is theorized to include

reactions involving singlet oxygen and amino acids with the light-excited dye [112]. Methylene blue and methylene green are two dyes that have been investigated for use in BHV fixation [109-112]. Investigation of multiple dyes has also been augmented by exploration of differing wavelengths of light as well as varying lengths of exposure [112].

In addition to demonstrating resistance to enzymatic degradation and calcification, tissues crosslinked using dye-mediated photooxidation have also exhibited low immunogenicity, non-cytotoxicity and seemingly improved physical properties [109-

112]. One particular fixation method, PhotoFix®_{, progressed to clinical trials but was cut}

short due to excessive regurgitation prior to two years. However, upon explantation, valves consistently exhibited signs of failure due to improper design while tissue performance seemed to be satisfactory [113]. Nonetheless, this has been a setback for the advancement of PhotoFix® and other alternative fixation chemistries; as the previously mentioned study suggests, current testing methods may be inadequate to properly predict clinical performance.

1.7.2. Carbodiimide Fixation

Water-soluble carbodiimides have also attracted a great deal of interest for use in alternative fixation methods. A popular pair of carbodiimide crosslinkers that have been investigated for use in BHV tissue fixation are 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). As mentioned previously, these reagents react to produce zero-length bonds within the tissue. This refers to the fact that neither EDC nor NHS remains in the bond after crosslink formation, but merely catalyzes the formation by utilizing available amino acid side groups [103,114-120].

Collagen crosslinking is initiated when EDC activates carboxyl groups on aspartic and glutamic acid, which then reacts with NHS to form a stable intermediate. NHS allows for increased crosslinking yield and facilitates reaction with nucleophiles, such as primary amines on lysine and hydroxylysine of collagen [114,115]. There is also evidence that these activated carboxyl groups can form ester crosslinks with hydroxyl groups, furthermore, in sufficient quantity to significantly affect tissue mechanics [114].

Tissue mechanics is a concern with carbodiimide crosslinked tissue as the zero- length bonds produced by crosslinks have been stated to yield undesirable collagen stiffness [116]. As such, some groups have investigated the incorporation of diamine spacer molecules into carbodiimide crosslinks that create “lengthened” bonds. These strategies aim to both enhance tissue stability and reduce stiffness, although, the majority of these alternative fixation studies have primarily focused on the reduction of calcification [116-119].

The amide bonds formed by EDC/NHS fixation are more stable than the Schiff- base bonds created by glutaraldehyde fixation, and it is theorized that carbodiimide crosslinking may offer superior collagen stability [117]. This theory is also suggested through a hypothesis submitted by Weadock et al., which proposes that improved stability is afforded due to the formation of a larger number of crosslinks using EDC/NHS treatment [115,121]. In addition to the collagen stability afforded by EDC/NHS fixation, GAGs can also be crosslinked through carbodiimide chemistry due to the high amount of available carboxyl groups situated on GAG molecules (Figure 3). The preservation of GAGs may further protect collagen sterically through the blockage of cleavage sites that are vulnerable to enzymatic attack [115]. GAGs may also improve

tissue stability through the improvement of tissue and valve biomechanics. Even slight improvements could delay the onset of fatigue damage and, as a result, slow structural degradation. Also, since EDC/NHS crosslinked tissues have exhibited resistance to calcification, carbodiimide crosslinking becomes an even more attractive alternative to glutaraldehyde fixation for improved BHV durability [116-119].

Figure 24. Representation of carbodiimide crosslinking in the presence of NHS [120].